The bi-functional protein 4a-carbinolamine dehydratase/dimerization cofactor of HNF-1 (DCoH) is involved in the regeneration of tetrahydrobiopterin, the cofactor of phenylalanine hydroxylase, and in the regulation of the transcription factor HNF-1. A mild form of hyperphenylalaninemia has been demonstrated to be caused by a defect in the enzymatic function and a decrease in proteolytic stability of DCoH. We have used site-directed mutagenesis to characterize the enzyme's catalytic core and to localize the site responsible for transcriptional coactivation.